Isolated rat pancreatic islets (A) or cells (B) were pre-incubated in low glucose for 2 hours and then stimulated with 16 mM glucose (glu), 100 nM insulin (ins), 60 mM K⁺ or 100 µM forskolin (fsk) for 1 hour. Isolated pancreatic islets (C) were pre-incubated in low glucose for 2 hours, pre-incubated in the presence or absence of 250 µM diazoxide (diaz) for 30 minutes and then stimulated with 16 mM glucose for 1 hour. Pancreatic islets or cells were homogenized and PDE3 activity was measured (n = 5–7 for A, n = 4 for B and n = 10 for C).

Recombinant ³²P-labelled PDE3B was immunoprecipitated and solubilized from crude membranes, run on SDS-PAGE, subjected to autoradiography and immunoblot analysis (one representative experiment shown). A. Stimulation with 16 mM glucose for 1 hour (n = 5). B. Stimulation with increasing glucose concentrations for 1 h. Quantification was made using a Fuji LAS 3000 Plus system.

A. Human islets were homogenized and total PDE, PDE1, PDE3 and PDE4 activity was measured (n = 3). B. 20–25 µg of islet homogenates from three different human donors were run on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were subjected to immunoblotting using PDE-isoform selective antibodies (one representative experiment shown).

A. INS-1 (832/13) cells were infected with either Ad-βgal or AdPDE3B virus for 2 hours, 16 hours prior to experiment. Cells were homogenized and analyzed by PDE3 activity assay or immunoblot analysis using anti-PDE3B antibody. B. Immunoisolation of recombinant ³²P-labelled PDE3B from AdPDE3B infected INS-1 (832/13) cells. A crude membrane fraction was used as starting material for immunoprecipitation using anti-PDE3B antibody or anti-flag M2 gel. Thoroughly washed immunoprecipitates were run on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were subjected to autoradiography, immunoblot analysis using anti-PDE3B antibodies and Ponceau S staining, respectively. C. Adβ-gal or AdPDE3B-infected INS-1 (832/13) cells were subjected to subcellular fractionation using density gradient centrifugation (Percoll™). Cytosol (Cyt), granule (Gran) and plasma membrane (PM) fractionations were prepared and analyzed with immunoblot analysis for PDE3B expression. The purity of the fractions was evaluated by immunoblot analysis of the plasma membrane marker Na⁺/K⁺-ATPase (n = 2). D. Cells were AdPDE3B-infected, pre-incubated in low glucose for 2 hours and then stimulated with 16 mM glucose, 100 nM insulin or 60 mM K⁺ for 1 hour. Cells were homogenized and PDE3 activity was measured (n = 3).

Immunoprecipitated recombinant ³²P-labelled PDE3B from crude membranes were run on SDS-PAGE, subjected to autoradiography and immunoblotting (one representative experiment shown). Quantification was made using Fuji LAS 3000 Plus system (n = 4 for A and n = 2 for B).